Abstract: 【Objective】To explore the effects of arsenic trioxide(ATO) on Warburg effect and Hedgehog signaling pathway of avian leukemia(AL)tumor cells,to clarify the mechanism of ATO alleviating AL liver tumor lesions in chickens,and to provide scientific basis for ATO treatment of AL in production.【Method】Acute toxicity test was carried out to determine the safety of ATO to green shell laying hens. The pathogen of avian leukemia virus(ALV) was identified by p27 antigen detection,PCR amplification and gp85 gene sequencing,and the pathological diagnosis was made by collec-ting the tumor tissue of infected chickens. AL chicken tumor cells were cultured in vitro and treated with 0.5,1.0 and 2.0 μmol/L ATO,respectively. The cells were collected after culture for 12,24 and 48 h,and the contents of glucose,lactic acid,hexokinase(HK)and glucose transporter 1(GLUT1) were detected,respectively. Green shell laying hens were artificially infected with ALV to construct a model group,and then treated with different doses of ATO(0.5,1.0 and 2.0 mg/kg·BW). ELISA was used to detect the protein levels of Hedgehog signaling pathway Gli1 and Shh in chicken liver tissue. The expression of Shh,Gli1,Gli2,Ptch2 and Smo genes was detected by real-time fluorescence quantitative PCR.【Result】The median lethal dose(LD₅₀) of ATO for green shell laying hens was 19.501 mg/kg·BW,and the LD₅₀-95% confidence limit was 19.501±0.213 mg/kg·BW. An ALV-J strain named GZCS01 was identified from green shell laying hens suspected to have AL,and the tumor formed by infection was nephroblastoma. At different doses of ATO(0.5,1.0 and 2.0 μmol/L),ATO was applied to wilms tumor cells. After 48 h culture,glucose content,lactate generation and HK contents in cell fluid were extremely significantly decreased(P<0.01,the same below),and GLUT1 content in wilms tumor cells was also decreased. Thus,the Warburg effect of tumor cells was weakened. High dose of ATO(2.0 mg/kg·BW) could extremely significantly or significantly inhibit the expression of Shh and Gli1 protein(P<0.05,the same below), extremely significantly or significantly inhibit the expression of Shh,Gli1,Gli2 and Smo genes,and extremely significantly up-regulate the expression of Ptch2 gene.【Conclusion】ATO can inhibit glucose uptake and lactic acid production of tumor cells,and reduce key enzyme production,thus weakening Warburg effect and inhibiting tumor cell proliferation. ATO can also reduce liver lesions in AL by down-regulating the expression of key factors in Hedgehog signaling pathway such as Shh,Gli1,Gli2 and Smo,and promoting the expression of Ptch2. It can be seen that the use of arsenic preparations(such as arsanic acid) in the treatment of AL is feasible.